Public health problem has always been a concern in the world. For example, hepatitis B virus and hepatitis C virus are the leading cause of morbidity of chronic hepatitis, and they directly lead to liver cancer. HIV causes AIDS infection, treponema pallidum causes sexually transmitted disease—syphilis, yet modern science and technology cannot control and treat these diseases well. In fact, whether the cancer or the infectious diseases, is one of the main threats to human health, this is mainly because of the lack of effective early diagnosis methods, corresponding preventive vaccines and effective drugs to treatment. Compared with the latter two, early diagnosis of disease may well be one of the most important means in the control of the development and dissemination of the diseases. From the perspective of prevention, laboratory diagnosis including traditional antigen-antibody reactions and the complex molecular biological diagnostic methods, these are all important effective methods for the diagnosis of diseases. Compared with the antigen-antibody detection, the molecular biological diagnosis based on biological macromolecules, mainly detects the genetic material of pathogenic (disease-causing) microorganisms—nucleotide, can shorten the window period of disease front several months to several days. The shortening of the window period is very important to the control of the spread of the diseases. Cur tend) the primary means of the detection of biomarkets in biological samples from infectious diseases (including nucleic acids and proteins), are enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) products and probe hybridization with gel imaging etc, of which enzyme linked immunosorbent assay (ELISA) belong to the detection means based on antigen-antibody reactions. From the body infected with pathogenic microorganisms to the produce of antibodies will take several months or even longer time, and this is almost equivalent to the time of the window period of the disease. The others are a variety of detection methods derived from the PCR-based technology. Although there are many advantages of these methods which detect the target genes in the biological samples, and these methods are major supplementary means or reconfirm “gold standard” means of clinical antigen-antibody detection methods at present. There exist the shortcomings of “false positive”, susceptible to pollution, high technical requirements, cumbersome and time-consuming etc. in this methods. These shortcomings are brought about by the PCR technology itself. Although these methods can detect at the molecular level, the rapid spread of infectious diseases made it a increasingly urgent need to address the problem to establish a low-cost, reliable, high sensitivity, high specificity, easy to operate, high-throughput DNA detection technology which can carry out diagnosis of a variety of diseases at the same time.
Nano-particles also known as ultrafine particles, generally refers to the particles in the size range from 1 to 100 nm, it is in the transition zone at the junction of atom clusters and macroscopic objects, and it is an excellent bio-molecular marker. The use of nano-probes which made from nano-materials in genetic testing methods, gradually attracts extensive attention in various fields of molecular diagnostics, etc., and increasingly demonstrates bright prospects for development.
In recent years, mass spectrometry (MS) technology has been developed, rapidly, and it has been widely used in the fields of chemical, petroleum, pharmaceutical, biotechnology, etc. it has become a very important tool in research and production. Especially the appearance of two “soft ionization” methods of matrix-assisted laser desorption ionization. (MALDI-TOF-MS, TOF MS) and electrospray ionization in the mid-1980s. with their characters of wide detection range, high sensitivity, easy to operate, high degree of automation and rapid detection, etc. they have been widely used in the study of biology, clinical medicine, environtology, etc. At present, German Qiagen Company has developed a set of technologies of encoding nucleic acids with organic molecules, and established a tag library of 64 encoded nucleic acid, which can simultaneously detect 22 pathogens with the use of mass spectrometry technology (Tgomas Briese et al. Diagnostic system for rapid and sensitive differential detection of pathogens. Emerging infectious diseases, 2005, 11(2), 310-313. Kokoris, Mark, et al. High-throughput SNP genotyping with the Masscode system. Molecular Diagnosis, 2000, 5(4), 329-340). This method binds the low molecular weight markers to nucleic acids with controllable optical switching technology, after PCR amplification, then conduct biometric identification, at last releases the markers by illumination and uses mass spectrometric detection to make judgments. This method designed very cleverly, but this method need bind the markers to biomolecules by chemical methods, and it need to release the markers again through chemical reactions before detection. This process is not only technically demanding, hut also cumbersome in operation and time-and-effort-consuming.
Patent CN101281164A disclosed an assembly method of mass-coded nano-probes, but the signal must be amplified by the PCR to read the detection sensitivity for the detection of the target. And it is not involved in the content of the practical applications, namely the durability test of this method of real biological samples. The specific process is: modify encoding molecules and corresponding recognition molecules to the surface of gold nano-particles, to make gold nano-probes. Bind gold nano-probes to target genes through hybridization, and then separate the nanoprobe-target complexes with treated silicon chip, at last detect the encoding molecules on the surface of colloidal gold-nanoprobes by mass spectrometric, thus the detection of target DNA can be achieved. Among which the encoding molecules are small organic molecules that can bind with gold nano-particles, and it can be detected by MS; and recognition molecules are DNA sequences that can carry out specific recognition reactions with target DNAs. The treated silicon chip has the role of capturing; gold-nanoprobe-target-DNA complexes, this is because that the surface of the silicon chip was bound with another DNA sequence which can carry out specific recognition reactions with target DNA and this DNA sequence is different from recognition molecules. This kind of modified silicon chip only has separation and purification effect, without concentration effect of the targets. This method realized signal encoding and amplification by nano-probes, and without the need of releasing the encoding molecules from nano-probes, it can detect the encoding molecules directly. The mass spectrometric detection of encoding molecules has a high degree of automation. The maximum of its sensibility is up to 10−14M. But the biggest flaw of the present patent is that it still needs to amplify the target DNA by PCR. The target genes can be detected unless its concentration was raised. The whole operation process is tedious, time consuming. And its sensitivity cannot reach the level required by clinical testing.